Microspheric bodies for use in screening therapies for Azheimer&#39;s disease and related conditions

ABSTRACT

Dense microspheres extracted and purified to homogeneity from whole brains and used in in vitro and in vivo screening tests for the detection of therapies effective in impeding amyloid formation and disease progression in human brain in Alzheimer&#39;s disease and related conditions.

This application is a continuation-in-part of application Ser. No.901,007 (filed Aug. 27, 1986), now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to microspheric bodies derived from brain cells.This invention also relates to the purification of these microsphericbodies to homogeneity and to their use in screening proposed therapeuticmeasures for effectiveness in impeding amyloid formation and diseaseprogression in human brain affected by Alzheimer's disease and relatedconditions. More specifically, the present invention is directed todense microspheres obtained in purified form from brain cells, to thepreparing of the dense microspheres in a usable form, and to methods forusing them in the identification of therapies for treating Alzheimer'sdisease and related conditions associated with the formation of amyloidfibrils in the brain.

Alzheimer's disease is an incurable brain disease affecting middle agedand elderly people throughout the world. According to most recentestimates, it is the 4th or 5th leading cause of death among NorthAmericans, and is responsible for inestimable personal and socialtragedy, loss of productivity, and custodial burden to society. There ispresently no widely-accepted effective treatment for Alzheimer'sdisease.

The principal symptom (manifestation) of Alzheimer's disease is the lossof higher mental faculties, typified by the loss of memory and behaviorreferred to as "dementia." Dementia is a symptom complex or syndromewhich can be seen in many brain diseases other than Alzheimer's disease,such as stroke, encephalitis and metabolic diseases. Since memory lossand dementia are relatively nonspecific symptoms, a certain and specificdefinition of Alzheimer's disease is based on the characteristicmicroscopic state of the brain, described initially by Marinesco,Alzheimer and others [see Alzheimer, A., Allgemeine Zeitschrift furPsychiatrie 64: 146-148, (1907); Marinesco, G., Comptes Rendus desSeances de la Socieete de Biologie et ses Filiales 70: 606-608 (1911)].

The particular microscopic feature that is a universally acceptedindicator of Alzheimer's disease, and that separates Alzheimer's diseasefrom other causes of dementia, is the accumulation of large numbers ofbrain lesions referred to as senile plaques and neurofibrillary tangles.These lesions (microscopical areas of abnormal brain tissue), when foundin suitable quantity in a brain sample, are the definitive criteria forthe diagnosis of Alzheimer's disease.

The clinical diagnosis of Alzheimer's disease is often a difficult andimperfect task which generally relies initially on ruling out othertreatable or clinically definable causes of dementia. In the appropriateclinical context, if the latter causes cannot be proven, Alzheimer'sdisease is often diagnosed antemortem, by exclusion, as the mostprobable diagnosis. Many indirect methods of diagnosis at present arebeing proposed and tested [see Conference Report, Khachaturian, Z. Arch.Neurol. 42: 1097-105 (1985)]; but the only certain and acceptable methodfor diagnosing Alzheimer's disease is by tissue microscopic histologicalstudy of a brain biopsy or necropsy sample, in which the above-mentionedsine qua non lesions are recognized by a certified specialist withadequate expertise.

Senile plaques in large quantities are essentially found only in theAlzheimer group of diseases; in contrast, neurofibrillary tangles arenonspecific, found in at least ten other neurological diseases [seeCorsellis, J.A.N., GREENFIELD's EUROPATHOLOGY 951-1025 (4th ed. 1984)(Edward Arnold, London)]. Individual senile plaques have roughly 1000Xthe volume of individual neurofibrillary tangles. True measurements oftotal brain senile plaque and neurofibrillary tangle content are notavailable, but on the above basis it is likely that the volume ofabnormal brain tissue occupied by senile plaques is many hundreds oftimes that of neurofibrillary tangles. The essential feature of thesenile plaque is the presence of amyloid fibrils ("amyloid"), which area congophilic red-green birefringent microfibrillar material (Corsellis,loc. cit.).

The utilization of materials found in human brain (normal or affected byAlzheimer's disease) that are not initially amyloid and transformingthem into amyloid has not been documented. Thus an experimental system,derived from human materials, characterized by the sine qua non featureof human Alzheimer's disease has not been documented. A fundamentalapproach to treating a disease is to reproduce the diseaseexperimentally in a nonhuman context and to test potential treatmentsfor their effect on the experimental disease. Because the presence ofamyloid provides a definitive indication of senile-plaque formation,most specialists agree that reproduction of amyloid fibrilsexperimentally from precursor materials which are extracted, activated,or otherwise derived from the human brain would constitute the bestavailable evidence linking an agent or precursor to the progression ofAlzheimer's disease. Despite much investigation into this questionduring the past fifty years, the fundamental step of reproducing amyloidexperimentally from materials derived solely from human brain tissue hasnot been documented.

A microscopic structure referred to as the dense microsphere is known toexist in normal brain and in brain affected by Alzheimer's disease brain(Averback, Acta Neuropathol. 61: 148-52 (1983); results confirmed byHara, M., J. Neuropath. Exp. Neurol. 1986). Some specialists believethat dense microspheres ("DMS") may be connected somehow to theformation of amyloid senile plaque, but this hypothessis has not beenproven. Evidence for the existence of DMS comes from microscopichistological section studies of fixed whole brain tissue, where thedense microspheres are observed as randomly dispersed, highly infrequentstructures occupying an estimated 10⁻⁹ or less of total brain volume, ata unit frequency roughly estimated at 10⁻¹⁶ or less, relative to otherdefinable brain structures such as mitochondria. But the extraction,purification and characterization of isolated samples, and the use oftangible DMS material to any advantage, have not been documented.Without these developments, DMS are structures of unproven function,unknown significance or usefulness, are not in tangible form and are notreadily available.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide densemicrospheres derived from brain cells in essentially homogeneous,purified form.

It is another object of the present invention to provide a method forscreening therapies for utility in impeding amyloid formation anddisease progression in human brains affected by Alzheimer's disease andrelated conditions.

In accomplising the foregoing objects, there has been provided, inaccordance with one aspect of the present invention, a composition ofmatter consisting essentially of dense microspheres derived frommammalian brain tissue, which bodies provide, when disrupted, materialthat displays congophilic birefringence.

In accordance with another aspect of the present invention, a method hasbeen provided for screening for the ability to impede amyloid formation,comprising the steps of (i) disrupting dense microspheres derived frommammalian brain tissue to form a material that is stainable with CongoRed stain; (ii) contacting the material with Congo Red stain and then(iii) subjecting the material to optical-microscopic examination todetect any congophilic birefringence. In a preferred embodiment, themethod of the present invention further comprises the step, prior tostep (i), of contacting the dense microspheres with a pharmacologicalagent, in order to ascertain the amyloid formation-impeding activity ofthat agent.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a micrographic representation of a DMS; and

FIG. 2 is another micrographic representation showing DMS in homogeneousform.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It has now been found to be most likely that, as a result of somepresently uncontrollable mechanism in the brain cells of certain livingbeings, DMS is disrupted to cause senile-plaque amyloid formation and,thereby, serious damage to the brain in the form of the lesions that arecharacteristic of Alzheimer's disease. The connection between DMSdisruption and amyloid formation is established by the fact thatdisrupted DMS treated with Congo Red stain display a red-greencongophilic birefringence identical to that found diagnostically inAlzheimer's disease senile-plaque amyloid.

In other words, the most significant aspect of the brain damage acceptedas characterizing Alzheimer's disease can, for the first and only knowntime, be induced and reproduced (according to established andconventional criteria) from and in material derived, pursuant to thepresent invention, exclusively from undamaged human brain samples.

The microspheric bodies employed in the present invention are derivedfrom mammalian brain tissue and are characterized, in essentiallyhomogeneous purified form, by a range of diameters from about 0.1 toabout 15 microns, by an outer membrane that surrounds proteinaceousmatter, and by certain stainability properties. For example, themicrospheric bodies according to the present invention are homogeneouslyelectron-dense when stained with osmium and lead, and can be visualizedby thin-section electron microscopy; under optical microscopicexamination, they appear eosinophilic and phloxinophilic, and arenonbirefringent when stained with Congo Red. When the microsphericbodies of the present invention are disrupted, a material is producedthat displays congophilic birefringence; that is, when stained withCongo Red the material becomes optically anisotropic to the extent ofsplitting an incident light wave into two waves with mutuallyperpendicular vibration directions.

As shown in FIG. 1, DMS are spherical, membrane-bounded, intracellularstructures, about 0.1 to 15 microns in diameter, that are found in humanand other mammalian brains. Homogeneous structures of DMS in purifiedfrom can be derived by extraction to give tangible samples ofhomogeneous globular bodies.

The following procedure can be followed to extract DMS from braintissue;

(1) Whole brain is removed from the skull postmortem, by use of standardpostmortem techniques for humans or animals. The best results areobtained if the organism has been in circulatory arrest for less thansix hours at the time of brain removal and if the body has beenrefrigerated as early as possible postmortem. DMS are still extractableat postmortem intervals greater than six hours and are still extractableif body cooling has been delayed or absent, but these two factors willusually greatly decrease the overall average yield of DMS per individualbrain. In addition to the effects of post-circulatory arrest intervaland temperature on DMS yield, there is considerable individual variationin DMS content per brain, and also individual variation in DMSextractability, which may be related to agonal metabolic state, overalldisease status or other factors. All of the factors which determinetotal DMS yield per brain can have an impact on DMS extraction, sincethe volume of purified sample of homogeneous DMS will decreaseproportionally to any reduction in percentage extractability; such adecrease may be sufficient to hinder accurate recognition during theextraction procedure. Furthermore, the screening of putative Alzheimer'sdisease therapies and the characterization of isolated samples of DMS,in accordance with the present invention, are rendered correspondinglymore difficult and costly, and ultimately may be impossible atcritically small volumes of DMS.

(2) By means of clean instruments, the freshly removed brain isimmediately dissected. Dissection is optimally performed in a cold roomat 10° C. By means of careful, but rapid, sharp and blunt dissection,the internal capsules, corona radiata, centra semi-ovale, brainstem,cerrebellum, lepto and pachymeninges, arachnoid granulations, choroidplexi, and blood vessels are separated and discarded, and the remainingmass of brain is rapidly used for the subsequent steps. (Standard blocksfor microscopic study can be removed at this stage and stored separatelyin histological fixative.) The dissected brain mass ("DBM") is optimallyutilized immediately after dissection. It may also be stored frozen attemperatures of -10° C. to -70° C, but this decreases the overallaverage yield of DMS per individual brain.

(3) The extraction of DSM material from DBM can be carried out by acombination of centrifugation steps. In an exemplary extraction, DBMmechanically homogenized in a 2:1 volume of 0.5M TRIS-HCL buffer (pH7.5) is subjected to centrifugation at about 200 rpm for some 10minutes. (All manipulations are carried out at around 4° C.) Thesediment thus obtained ("Sediment I") is separated across a sucrosegradient (1.589M, or 45%; 1.895M, or 52%; 2.3895M, or 62.5%) viacentrifugation at 26,000 rpm for 30 minutes. It has been found that thematerial that settles at the interface between 1.895M and 2.1015M (56.7%sucrose) is the DMS-containing fraction, as may be confirmed bymicroscopic examination, with eosin staining, of the fraction.

The DMS-containing fraction obtained from Sediment I consistsessentially of the dense microspheres described above, and can be usedin a screening method according to the present invention. It ispreferable, however, for the fraction to be subjected to additionalmanipulations in order to enrich the DMS concentration. To this end, ithas proved useful, for example, to wash the DMS-containing fraction inbuffer--the above-mentioned homogenization buffer is suitable for thispurpose--nd to spin the resulting mixture again (10,000 rpm for 7minutes) to obtain DMS-enriched sediment ("Sediment II").

As with Sediment I, Sediment II can be run through a density gradient toenrich further the yield of DMS. It has been discovered that thecarbohydrate Percoll® (Pharmacia) is particularly useful in thiscontext. A commercially available formulation of 80% Percoll (1.13 g/ml)in 0.15M NaCl provides a isoosmolar gradient to which Sediment II can besubjected (30,000 rpm for 15 minutes); successive samples, say, on theorder of 0.25 to 1 cc each, can then be taken along the length of thegradient and the DMS-enriched fractions isolated. After these fractionsare washed again in buffer, they can be spun down once again (15,000 rpmfor 10 minutes) to obtain a sediment ("Sediment III") that issubstantially pure DMS.

The DMS materials obtained as described above can be used, pursuant tothe present invention, in screening Alzheimer's disease therapies. Morespecifically, a DMS material within the present invention can beemployed to ascertain effectiveness, on the part of an active agent ortreatment comprising a putative Alzheimer's therapy, in preventing thered-to-green congophilic birefringence that accompanies the formation ofamyloid when DMS are disrupted. By whatever means DMS are disrupted incontrol samples, a proposed therapy can be screened by virtue of itsability to retard or preclude amyloid formation under test conditions.

For example, in vitro DMS disruption on an appropriate viewing surface,such as a glass or plastic slide (see Test 1 below), can be accomplishedby mechanical means; by the action of an enzyme treatment, as in a 10%trypsin or pepsin solution, or other chemical exposure, as to a 10%guanine solution; or by exposing DMS material to extreme pH values (atroom temperature, pH2 or pH10) or temperatures (e.g., 100° C. for onehour). Disruption of DMS can also be effected by injecting DMS materialof the present invention into an isolated tissue sample (see Test 2);brain slices are preferred for this purpose, but liver, pancreas andother organs are also acceptable sources for tissue samples.

Alternatively, DMS can be disrupted in vivo, by injecting the purifiedDMS material of the present invention into a suitable laboratory animal.Since simple injection of DMS onto a glass slide does not result inamyloid formation, it is understood that DMS disruption (and productionof amyloid) in vivo, as reported in Example 2 below, occurs in theextracellular spaces of the injected tissue. Although brain sites forinjection are preferred, injection sites elsewhere in a test animal'sbody, such as in skin and in muscle, are suitable for determining theability of a proposed active agent or treatment step to hinder theresulting formation of amyloid.

The following test paradigms illustrate various ways in which DMSmaterial, as described above, can be used according to the presentinvention to screen potential Alzheimer therapies.

TEST 1. In vitro disruption of DMS on a glass slide.

Homogeneous DMS preparations are placed by droplet on a clean dry glassslide. The volume and number of DMS used is optional but is recommendedto be at least several thousand to facilitate interpretation (seeExample 1 below). Larger samples are more costly but are easier and moreunequivocal to examine. The DMS are mechanically disrupted using astainless steel spatula scraping and pressing the DMS against a glassslide in repetitive manual back and forth motions for one minute.

The slide is allowed to air dry. A few drops of Congo Red stain are thenadded to the dried slide and gently passed over the dried disrupted DMSfor 30 seconds and the stain is then drained off the slide onto tissuepaper or filter paper. The slide is then examined in the lightmicroscope, the latter fitted with crossed polarizing lenses to assessred-green congophilic birefringence. The result is an unequivocalred-to-green ("apple green") birefringence similar to the red-to-applegreen birefringence found in the senile-plaque amyloid of Alzheimerpatients, and in quantities proportional to the volume of DMS applied tothe slide initially. All other reactions, staining results, orquantitatively insignificant results are considered negative in theabsence of the characteristic color change-positive staining result, inquantity proportional to the volume of DMS applied, which indicates thatdisrupted DMS are of the nature of senile-plaque amyloid.

For test purposes, a corresponding DMS sample is contacted with apossible pharmacological agent, and the DMS disruption/stainingprocedure as described above is repeated. Many variations are possible,e.g., the active agent may be applied to the DMS in solution, beforeapplication to the slide, or to the DMS on the slide. In any event, anobservation that the agent prevents development of the red-greenbirefringence in the above-described negative control slide (no agentpresent) is a clear indication that the active agent should be testedfurther for efficacy against Alzheimer's disease.

As a positive control, the test slide can also be compared to a slideupon which DMS were disrupted after contact with a 1% aqueous solutionof diphenyl disazo binaphthionic acid, [C₆ H₄ N₂ C₁₀ H₅ [NH₂ ]SO₂ ONa]₂; the compound, Congo Red, is described by Graves & Kickham, New EnglandJ. Med. 214: 782-83 (1936), and Wallace, The Lancet (Feb. 20, 1932), at391-393, an has been found to block amyloid formation in the presentinvention.

TEST 2. In vitro disruption of DMS in a human brain slice.

Human brain postmortem samples of histological block size (block size iselective; usually 1-tcm ×1-5 cm×1-3mm) are removed, by steriletechniques, with the aid of sterile gloves, scalpel and forceps, andthen are placed in sterile empty plastic containers, such as a Petridish before extracted DMS are injected into each brain sample at roomtemperature. After one hour incubation at room temperature, the brainsamples are immersed in situ in histological fixative and processed forhistology by techniques that are standard for optical microscopy.Controls, size of inoculum, preparations of slides and interpretation ofresults are covered under discussion of in vivo Test 3 below.

TEST 3. In vivo disruption of DMS by injection into live tissue.

Laboratory rodents are anesthetized and their brains immobilized byroutine methods and injections of purified DMS are made into superficialcerebral cortex regions through sterile needles inserted through theskull and meninges. (Sham control injections of DMS negative materialcan be put into either the contralateral cortex or into separateanimals.) The method of anesthesia, type of craniotomy, site ofinjections in the brain parenchyma, size of needle and syringe or othervehicle, wound closure technique, and numbers of animals used are notcrucial to the test and will vary depending on the animal used. Thus, asmall mouse may not need a skull flap whereas a larger mammal may need aburr hole; size of needles or vehicles may vary with animal brain size,etc. (see Example 1). The size of injection is elective: smallerinjections are more difficult and costly to trace histologically (seebelow), but larger injections are more costly in terms of numbers of DMSused. (A sample protocol is detailed in Example 1.) The animal ispainlessly sacrificed 0.5 hours or more after injection. The exact timeof sacrifice is elective: generally 1-24 hours is preferable but the DMStransformation will persist and can be recognized at many timeintervals. After sacrifice the brain is removed by standard methods andimmersion fixed in histological fixative. Perfusion fixation is notrecommended because perfusion pressures will usually disrupt theinjection cavity and render the results inaccessible. According tostandard methods, the brain is fixed in toto for several days(correspondingly longer for larger animal brains), sliced, embedded,cut, mounted and stained for histological study. A dissecting microscopeis used to locate the injection site and accurately place it in theblock, and sections are carefully inspected during microtomy to ensurethat the injection site is in the section and not discarded duringtrimming. the mounted slides are stained with Congo Red according to thestandard method. The sections are examined with the optical microscopefitted with polarizing lenses as above and assessed as described abovewith regard to the in vitro test.

The use of positive and negative controls, and the testing of putativeactive agents, are carried out in a manner analogous to that followedthe in vitro tests described above, with further variations possible byvirtue of the fact that test compounds can be are tested in vivo viainjection, ingestion and/or other possible routes, before, after orduring the introduction of DMS, with or without the DMS. In addition,therapeutic strategies other than those based on the action of apharmacological agent can be studied in whole animals.

By means of the above tests, nontoxic compounds suitable for clinicaltesting in human beings can be found, pursuant to the present invention,that block the transformation of dense microspheres into amyloid.Because it is immunogenic in standard laboratory animals, the DMSmaterial of the present invention can also be used to produce polyclonaland monoclonal antibodies against dense microspheres. These antibodies,in turn, can be employed in ELISA-type assays, see, e.g., VOLLER et al,THE ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) (Dynatech Laboratories1979), and other immunological tests, such as radioimmunoassays, fordetecting DMS in biological samples. Via conventional techniques, asdescribed, for example, by Kennet et al, Curr. Top. Microbiol. Immunol.81: 77-91 (1978), anti-DMS antibodies can be produced using the DMSmaterial of the present invention and then "tagged" with a radionuclide,a colorimetric agent or a fluorescent marker. The tagged antibodies canbe used in diagnostic tests to detect the presence of the densemicrospheres with which the antibodies react, rendering the microspheresvisualizable.

Other details of the present invention are further described byreference to the following illustrative examples.

EXAMPLE 1

Approximately 80,000 homogeneously extracted DMS were placed in adroplet from a Pasteur pipette onto a glass slide, and the droplet wasair dried. The DMS adhered to the glass slide. Acetylcholine (20 mM) inphysiological saline was added in a droplet, as a possible testcompound, to DMS. The DMS were mechanically disrupted, in the presenceof the test compound, as described above and then were allowed to airdry. A control slide was treated exactly in the same manner, except thatthe test compound was not added. Congo Red stain was added, and thereaction products were examined, as previously described. Underoptical-microscopic examination with a polarizing lens, both slidesshowed abundant reddish stained material, respectively, in quantities ofthe order of magnitude as in the initial DMS droplet quantity. Thereddish material was observed to turn a brilliant apple green duringrotation of the polarizing lens. Rotation of the polarizing lenses backand forth demonstrated unequivocal and abundant red-to-green andgreen-to-red birefringence which persisted indefinitely. That bothslides showed these indistinguishable results meant that acetylcholinehad tested negative, i.e., had showed no useful activity in this test.

EXAMPLE 2

Four male Wistar rats of 3 months age were anesthetized by etherinhalation. Their heads are immobilized by means of a stereotactic headbrace. Bilateral parieto-occipital scalp incisions (1 cm) were made witha sterile scalpel blade. Bilateral parieto-occipital 0.5 mm burr holesare made with a 0.5 mm drill. In two rats 400,000 human DMS are injectedon one side through the burr hole from a sterile 1 cc syringe fittedwith a sterile 22 gauge needle. The injection was made into the cerebralcortex to a depth of a few mm so that the injection was within theparenchyma, not on the surface and not in the ventricles. On thecontralateral side, 100 μL of homogenized whole human brain wereinjected. In two rats, the DMS as above were injected on one side, whileon the contralateral side 100 μL of sterile physiological saline wereinjected. One rat from each group had been premedicated with a testcompound; the other rat had not been treated.

After injection, a sterile suture was placed through the scalp incisionto cover the wound, and the animals are observed for one hour. After onehour the animals were painlessly sacrificed by ether inhalation and CO₂insufflation. The brains were removed and processed, and the resultsinterpreted, as described above, with the result that the test compoundwas found to be effective in preventing formation of amyloid senileplaque.

What is claimed is:
 1. A composition of matter consisting essentially ofa suspension of dense microspheres in a liquid carrier, said densemicrospheres in said liquid carrier being derived from mammalian braintissue and providing, when disrupted, material that displays congophilicbirefringence.
 2. A composition according to claim 1, wherein saidmicrospheres are present in said suspension in essentially homogeneous,purified form.
 3. A composition according to claim 1, wherein saidliquid carrier is a buffer solution.
 4. A composition that is theproduct of a process comprising the steps of (A) subjecting homogenizedmammalian brain to a first centrifugation, at about 200 rpm for about 10minutes, to obtain a sediment; (B) separating said sediment across asucrose gradient via a second centrifugation at about 26,000 rpm forabout 30 minutes, said sucrose gradient comprising a variation insucrose concentration between at least a first level of about 52%sucrose content and a second level of about 56.7% sucrose content andfurther comprising an interface between said first level and said secondlevel; and (C) isolating material that settles at the interface betweensaid first level and said second level of said gradient, said materialcontaining dense microspheres.
 5. A composition that is the product of aprocess comprising the steps of (A) subjecting homogenized mammalianbrain to a first centrifugation, at about 200 rpm for about 10 minutes,to obtain a sediment; (B) separating said sediment across a sucrosegradient via a second centrifugation at about 26,000 rpm for about 30minutes, said sucrose gradient comprising a variation in sucroseconcentration between at least a first level of about 52% sucrosecontent and a second level of about 56.7% sucrose content and furthercomprising an interface between said first level and said second level;(C) isolating material that settles at the interface between said firstlevel and said second level of said gradient, said material containingdense microspheres; and (D) enriching said material as to theconcentration of said dense microspheres to obtain amicrosphere-enriched material.
 6. A product according to claim 5,wherein said step of enriching said material comprises subjecting saidmaterial to a third centrifugation, at about 10,000 rpm for about 7minutes, to obtain a microsphere-enriched sediment.
 7. A compositionthat is the product of a process comprising the steps of (A) subjectinghomogenized mammalian brain to a first centrifugation, at about 200 rpmfor about 10 minutes, to obtain a sediment; (B) separating said sedimentacross a sucrose gradient via a second centrifugation at about 26,000rpm for about 30 minutes, said sucrose gradient comprising a variationin sucrose concentration between at least a first level of about 52%sucrose content and a second level of about 56.7% sucrose content andfurther comprising an interface between said first level and said secondlevel; (C) isolating material that settles at the interface between saidfirst level and said second level of said gradient, said materialcontaining dense microspheres; (D) enriching said material as to theconcentration of said dense microspheres by subjecting said material toa third centrifugation, at about 10,000 rpm for about 7 minutes, toobtain a microsphere-enriched sediment; and (E) separating saidmicrosphere-enriched sediment over a density gradient and obtaining amicrosphere-enriched fraction of said density gradient.